Figure 4.
The RRMs as well as the RGG/RG motif contribute to the RNA annealing activity of AUF1 p45. (a) Scheme of the fluorescence-based 5ʹCS-3ʹCS RNA annealing assay to analyze the hybridization of the CS cyclization sequences. Annealing of the complementary 5′- and 3′CS RNAs that are fluorescently labeled with Cy5 and Cy3, respectively, leads to a detectable FRET signal. (b) Example kinetic traces of the RNA-RNA interaction in the presence of the indicated concentrations of AUF1 p45 or variant RRM2mut. Cy3-labeled 3′CS RNA was incubated with the respective protein, followed by the addition of Cy5-labeled 5′CS RNA. The fluorescence signals were analyzed according to a second-order reaction Equation (3). (c) – (e) The observed rate constants kobs (s−1) that were measured for the RNA-RNA interaction reaction in the presence of AUF1 p45 or variants thereof were plotted as a function of the protein concentration.